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OBJECTIVE: To investigate whether periosteal cells from adult humans have features of multipotent mesenchymal stem cells (MSCs) at the single-cell level. METHODS: Cell populations were enzymatically released from the periosteum of the proximal tibia obtained from adult human donors and then expanded in monolayer. Single-cell-derived clonal populations were obtained by limiting dilution. Culture-expanded periosteal cell populations were tested for their growth potential and for expression of conventional markers of MSCs and were subjected to in vitro assays to investigate their multilineage potential. To assess their multipotency in vivo, periosteal cells were injected into a regenerating mouse tibialis anterior muscle for skeletal myogenesis or were either seeded into an osteoinductive matrix and implanted subcutaneously into nude mice for osteogenesis or implanted in a joint surface defect under a periosteal flap into goats for chondrogenesis. Cell phenotypes were analyzed by histochemistry and immunohistochemistry and by reverse transcription-polymerase chain reaction for the expression of lineage-related marker genes. RESULTS: Regardless of donor age, periosteal cells were clonogenic and could be expanded extensively in monolayer, maintaining linear growth curves over at least 30 population doublings. They displayed long telomeres and expressed markers of MSCs. Under specific conditions, both parental and single-cell-derived clonal cell populations differentiated to the chondrocyte, osteoblast, adipocyte, and skeletal myocyte lineages in vitro and in vivo. CONCLUSION: Our study demonstrates that, regardless of donor age, the adult human periosteum contains cells that, upon enzymatic release and culture expansion, are multipotent MSCs at the single-cell level. [ABSTRACT FROM AUTHOR]

In brain metastasis, cancer cells remain in close contact with the existing vasculature and can use vessels as migratory paths-a process known as vessel co-option. However, the mechanisms regulating this form of migration are poorly understood. Here we use ex vivo brain slices and an organotypic in vitro model for vessel co-option to show that cancer cell invasion along brain vasculature is driven by the difference in stiffness between vessels and the brain parenchyma. Imaging analysis indicated that cells move along the basal surface of vessels by adhering to the basement membrane extracellular matrix. We further show that vessel co-option is enhanced by both the stiffness of brain vasculature, which reinforces focal adhesions through a talin-dependent mechanism, and the softness of the surrounding environment that permits cellular movement. Our work reveals a mechanosensing mechanism that guides cell migration in response to the tissue's intrinsic mechanical heterogeneity, with implications in cancer invasion and metastasis., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Hypertrophic cardiomyopathy (HCM) is characterized by thickening of the left ventricular wall, diastolic dysfunction, and fibrosis, and is associated with mutations in genes encoding sarcomere proteins. While in vitro studies have used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study HCM, these models have not examined the multicellular interactions involved in fibrosis. Using engineered cardiac microtissues (CMTs) composed of HCM-causing MYH7 -variant hiPSC-CMs and wild-type fibroblasts, we observed cell-cell cross-talk leading to increased collagen deposition, tissue stiffening, and decreased contractility dependent on fibroblast proliferation. hiPSC-CM conditioned media and single-nucleus RNA sequencing data suggested that fibroblast proliferation is mediated by paracrine signals from MYH7 -variant cardiomyocytes. Furthermore, inhibiting epidermal growth factor receptor tyrosine kinase with erlotinib hydrochloride attenuated stromal activation. Last, HCM-causing MYBPC3 -variant CMTs also demonstrated increased stromal activation and reduced contractility, but with distinct characteristics. Together, these findings establish a paracrine-mediated cross-talk potentially responsible for fibrotic changes observed in HCM.

Generating stable and customizable topography on hydrogel surfaces with contact guidance potential is critical as it can direct/influence cell growth. This necessitates the development of new techniques for surface patterning of the hydrogels. We report on the design of a square grid template for surface patterning hydrogels. The template was 3-D printed and has the diameter of a well in a 24-well plate. Hyaluronic acid methacrylate (HA) hydrogel precursor solutions were cast on the 3D printed template's surface, which generated 3D square shape topographies on the HA hydrogel surface upon demolding. The 3D Laser Microscopy has shown the formation of a periodic array of 3D topographies on hydrogel surfaces. 3D Laser and Electron Microscopy Imaging have revealed that this new method has increased the surface area and exposed the underlying pore structure of the HA hydrogels. To demonstrate the method's versatility, we have successfully applied this technique to generate 3D topography on two more acrylate hydrogel formulations, gelatin Methacrylate and polyethylene glycol dimethacrylate. Human neonatal dermal fibroblast cells were used as a model cell line to evaluate the cell guidance potential of patterned HA hydrogel. Confocal fluorescence microscopy imaging has revealed that the 3D surface topographies on HA hydrogels can guide and align the actin filaments of the fibroblasts presumably due to the contact guidance mechanism. The newly developed methodology of 3D topography generation in acrylate hydrogels may influence the cell responses on hydrogel surfaces which can impact biomedical applications such as tissue engineering, wound healing, and disease modeling., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Previous lung-on-chip devices have facilitated significant advances in our understanding of lung biology and pathology. Here, we describe a novel lung-on-a-chip model in which human induced pluripotent stem cell-derived alveolar epithelial type II cells (iAT2s) form polarized duct-like lumens alongside engineered perfused vessels lined with human umbilical vein endothelium, all within a 3D, physiologically relevant microenvironment. Using this model, we investigated the morphologic and signaling consequences of the KRAS G12D mutation, a commonly identified oncogene in human lung adenocarcinoma (LUAD). We show that expression of the mutant KRAS G12D isoform in iAT2s leads to a hyperproliferative response and morphologic dysregulation in the epithelial monolayer. Interestingly, the mutant epithelia also drive an angiogenic response in the adjacent vasculature that is mediated by enhanced secretion of the pro-angiogenic factor soluble uPAR. These results demonstrate the functionality of a multi-cellular in vitro platform capable of modeling mutation-specific behavioral and signaling changes associated with lung adenocarcinoma., Competing Interests: C.S.C. is a founder and owns shares of Innolign Biomedical, a company that is developing engineered organ models for pharmaceutical research and development, and Satellite Biosciences, a company that is developing cell-based therapies. The other authors declare that they have no competing interest., (© 2024 Author(s).)

Tissue engineering has long sought to rapidly generate perfusable vascularized tissues with vessel sizes spanning those seen in humans. Current techniques such as biological 3D printing (top-down) and cellular self-assembly (bottom-up) are resource intensive and have not overcome the inherent tradeoff between vessel resolution and assembly time, limiting their utility and scalability for engineering tissues. We present a flexible and scalable technique termed SPAN - S acrificial P ercolation of A nisotropic N etworks, where a network of perfusable channels is created throughout a tissue in minutes, irrespective of its size. Conduits with length scales spanning arterioles to capillaries are generated using pipettable alginate fibers that interconnect above a percolation density threshold and are then degraded within constructs of arbitrary size and shape. SPAN is readily used within common tissue engineering processes, can be used to generate endothelial cell-lined vasculature in a multi-cell type construct, and paves the way for rapid assembly of perfusable tissues., Competing Interests: DECLARATION OF INTERESTS A provisional patent application has been filed by Boston University based on this work. C.S.C. is a founder and owns shares of Innolign Biomedical, a company that is developing engineered organ models for pharmaceutical research and development; Satellite Biosciences, a company that is developing cell-based therapies; and Ropirio Therapeutics, a company developing molecular therapeutics for vascular disease. The other authors declare that they have no competing interests.

Although tissue culture plastic has been widely employed for cell culture, the rigidity of plastic is not physiologic. Softer hydrogels used to culture cells have not been widely adopted in part because coupling chemistries are required to covalently capture extracellular matrix (ECM) proteins and support cell adhesion. To create an in vitro system with tunable stiffnesses that readily adsorbs ECM proteins for cell culture, we present a novel hydrophobic hydrogel system via chemically converting hydroxyl residues on the dextran backbone to methacrylate groups, thereby transforming non-protein adhesive, hydrophilic dextran to highly protein adsorbent substrates. Increasing methacrylate functionality increases the hydrophobicity in the resulting hydrogels and enhances ECM protein adsorption without additional chemical reactions. These hydrophobic hydrogels permit facile and tunable modulation of substrate stiffness independent of hydrophobicity or ECM coatings. Using this approach, we show that substrate stiffness and ECM adsorption work together to affect cell morphology and proliferation, but the strengths of these effects vary in different cell types. Furthermore, we reveal that stiffness mediated differentiation of dermal fibroblasts into myofibroblasts is modulated by the substrate ECM. Our material system demonstrates remarkable simplicity and flexibility to tune ECM coatings and substrate stiffness and study their effects on cell function., Competing Interests: Conflict of Interest C.S.C. is a founder and own shares of Innolign Biomedical, a company that is developing engineered organ models for pharmaceutical research and development, and Satellite Biosciences, a company that is developing cell-based therapies. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Impaired lymphatic drainage and lymphedema are major morbidities whose mechanisms have remained obscure. To study lymphatic drainage and its impairment, we engineered a microfluidic culture model of lymphatic vessels draining interstitial fluid. This lymphatic drainage-on-chip revealed that inflammatory cytokines that are known to disrupt blood vessel junctions instead tightened lymphatic cell-cell junctions and impeded lymphatic drainage. This opposing response was further demonstrated when inhibition of rho-associated protein kinase (ROCK) was found to normalize fluid drainage under cytokine challenge by simultaneously loosening lymphatic junctions and tightening blood vessel junctions. Studies also revealed a previously undescribed shift in ROCK isoforms in lymphatic endothelial cells, wherein a ROCK2/junctional adhesion molecule-A (JAM-A) complex emerges that is responsible for the cytokine-induced lymphatic junction zippering. To validate these in vitro findings, we further demonstrated in a genetic mouse model that lymphatic-specific knockout of ROCK2 reversed lymphedema in vivo. These studies provide a unique platform to generate interstitial fluid pressure and measure the drainage of interstitial fluid into lymphatics and reveal a previously unappreciated ROCK2-mediated mechanism in regulating lymphatic drainage.

Competing Interests: Declaration of interests The author declares no competing interests.

Although the mechanisms underlying wound healing are largely preserved across wound types, the method of injury can affect the healing process. For example, burn wounds are more likely to undergo hypertrophic scarring than are lacerations, perhaps due to the increased underlying damage that needs to be cleared. This tissue clearance is thought to be mainly managed by immune cells, but it is unclear if fibroblasts contribute to this process. Herein, we utilize a 3D in vitro model of stromal wound healing to investigate the differences between two modes of injury: laceration and laser ablation. We demonstrate that laser ablation creates a ring of damaged tissue around the wound that is cleared by fibroblasts prior to wound closure. This process is dependent on ROCK and dynamin activity, suggesting a phagocytic or endocytic process. Transmission electron microscopy of fibroblasts that have entered the wound area reveals large intracellular vacuoles containing fibrillar extracellular matrix. These results demonstrate a new model to study matrix clearance by fibroblasts in a 3D soft tissue. Because aberrant wound healing is thought to be caused by an imbalance between matrix degradation and production, this model, which captures both aspects, will be a valuable addition to the study of wound healing., (© 2023 Author(s).)

After a myocardial infarction (MI), the heart undergoes changes including local remodeling that can lead to regional abnormalities in mechanical and electrical properties, ultimately increasing the risk of arrhythmias and heart failure. Although these responses have been successfully recapitulated in animal models of MI, local changes in tissue and cell-level mechanics caused by MI remain difficult to study in vivo. Here, we developed an in vitro cardiac microtissue (CMT) injury system that through acute focal injury recapitulates aspects of the regional responses seen following an MI. With a pulsed laser, cell death was induced in the center of the microtissue causing a loss of calcium signaling and a complete loss of contractile function in the injured region and resulting in a 39% reduction in the CMT's overall force production. After 7 days, the injured area remained void of cardiomyocytes (CMs) and showed increased expression of vimentin and fibronectin, two markers for fibrotic remodeling. Interestingly, although the injured region showed minimal recovery, calcium amplitudes in uninjured regions returned to levels comparable with control. Furthermore, overall force production returned to preinjury levels despite the lack of contractile function in the injured region. Instead, uninjured regions exhibited elevated contractile function, compensating for the loss of function in the injured region, drawing parallels to changes in tissue-level mechanics seen in vivo. Overall, this work presents a new in vitro model to study cardiac tissue remodeling and electromechanical changes after injury. NEW & NOTEWORTHY We report an in vitro cardiac injury model that uses a high-powered laser to induce regional cell death and a focal fibrotic response within a human-engineered cardiac microtissue. The model captures the effects of acute injury on tissue response, remodeling, and electromechanical recovery in both the damaged region and surrounding healthy tissue, modeling similar changes to contractile function observed in vivo following myocardial infarction.

Biomimetic on-chip tissue models serve as a powerful tool for studying human physiology and developing therapeutics; however, their modeling power is hindered by our inability to develop highly ordered functional structures in small length scales. Here, we demonstrate how high-precision fabrication can enable scaled-down modeling of organ-level cardiac mechanical function. We use two-photon direct laser writing (TPDLW) to fabricate a nanoscale-resolution metamaterial scaffold with fine-tuned mechanical properties to support the formation and cyclic contraction of a miniaturized, induced pluripotent stem cell-derived ventricular chamber. Furthermore, we fabricate microfluidic valves with extreme sensitivity to rectify the flow generated by the ventricular chamber. The integrated microfluidic system recapitulates the ventricular fluidic function and exhibits a complete pressure-volume loop with isovolumetric phases. Together, our results demonstrate a previously unexplored application of high-precision fabrication that can be generalized to expand the accessible spectrum of organ-on-a-chip models toward structurally and biomechanically sophisticated tissue systems.

The Notch pathway regulates complex patterning events in many species and is critical for the proper formation and function of the vasculature. Despite this importance, how the various components of the Notch pathway work in concert is still not well understood. For example, NOTCH1 stabilizes homotypic endothelial junctions, but the role of NOTCH1 in heterotypic interactions is not entirely clear. NOTCH3, on the other hand, is essential for heterotypic interactions of pericytes with the endothelium, but how NOTCH3 signaling in pericytes impacts the endothelium remains elusive. Here, we use in vitro vascular models to investigate whether pericyte-induced stabilization of the vasculature requires the cooperation of NOTCH1 and NOTCH3. We observe that both pericyte NOTCH3 and endothelial NOTCH1 are required for the stabilization of the endothelium. Loss of either NOTCH3 or NOTCH1 decreases the accumulation of VE-cadherin at endothelial adherens junctions and increases the frequency of wider, more motile junctions. We found that DLL4 was the key ligand for simulating NOTCH1 activation in endothelial cells and observed that DLL4 expression in pericytes is dependent on NOTCH3. Altogether, these data suggest that an interplay between pericyte NOTCH3 and endothelial NOTCH1 is critical for pericyte-induced vascular stabilization.

Gap closure is a dynamic process in wound healing, in which a wound contracts and a provisional matrix is laid down, to restore structural integrity to injured tissues. The efficiency of wound closure has been found to depend on the shape of a wound, and this shape dependence has been echoed in various in vitro studies. While wound shape itself appears to contribute to this effect, it remains unclear whether the alignment of the surrounding extracellular matrix (ECM) may also contribute. In this study, we investigate the role both wound curvature and ECM alignment have on gap closure in a 3D culture model of fibrous tissue. Using microfabricated flexible micropillars positioned in rectangular and octagonal arrangements, seeded 3T3 fibroblasts embedded in a collagen matrix formed microtissues with different ECM alignments. Wounding these microtissues with a microsurgical knife resulted in wounds with different shapes and curvatures that closed at different rates. Observing different regions around the wounds, we noted local wound curvature did not impact the rate of production of provisional fibronectin matrix assembled by the fibroblasts. Instead, the rate of provisional matrix assembly was lowest emerging from regions of high fibronectin alignment and highest in the areas of low matrix alignment. Our data suggest that the underlying ECM structure affects the shape of the wound as well as the ability of fibroblasts to build provisional matrix, an important step in the process of tissue closure and restoration of tissue architecture. The study highlights an important interplay between ECM alignment, wound shape, and tissue healing that has not been previously recognized and may inform approaches to engineer tissues. Impact statement Current models of tissue growth have identified a role for curvature in driving provisional matrix assembly. However, most tissue repair occurs in fibrous tissues with different levels of extracellular matrix (ECM) alignment. Here, we show how this underlying ECM alignment may affect the ability of fibroblasts to build new provisional matrix, with implications for in vivo wound healing and providing insight for engineering of new tissues.

Progressive loss of cardiac systolic function in arrhythmogenic cardiomyopathy (ACM) has recently gained attention as an important clinical consideration in managing the disease. However, the mechanisms leading to reduction in cardiac contractility are poorly defined. Here, we use CRISPR gene editing to generate human induced pluripotent stem cells (iPSCs) that harbor plakophilin-2 truncating variants ( PKP2 tv), the most prevalent ACM-linked mutations. The PKP2 tv iPSC–derived cardiomyocytes are shown to have aberrant action potentials and reduced systolic function in cardiac microtissues, recapitulating both the electrical and mechanical pathologies reported in ACM. By combining cell micropatterning with traction force microscopy and live imaging, we found that PKP2 tvs impair cardiac tissue contractility by destabilizing cell-cell junctions and in turn disrupting sarcomere stability and organization. These findings highlight the interplay between cell-cell adhesions and sarcomeres required for stabilizing cardiomyocyte structure and function and suggest fundamental pathogenic mechanisms that may be shared among different types of cardiomyopathies.

How adhesive forces are transduced and integrated into biochemical signals at focal adhesions (FAs) is poorly understood. Using cells adhering to deformable micropillar arrays, we demonstrate that traction force and FAK localization as well as traction force and Y397-FAK phosphorylation are linearly coupled at individual FAs on stiff, but not soft, substrates. Similarly, FAK phosphorylation increases linearly with external forces applied to FAs using magnetic beads. This mechanosignaling coupling requires actomyosin contractility, talin-FAK binding, and full-length vinculin that binds talin and actin. Using an in vitro 3D biomimetic wound healing model, we show that force-FAK signaling coupling coordinates cell migration and tissue-scale forces to promote microtissue repair. A simple kinetic binding model of talin-FAK interactions under force can recapitulate the experimental observations. This study provides insights on how talin and vinculin convert forces into FAK signaling events regulating cell migration and tissue repair.